CN112430680B - Specific DNA molecular marker for sex identification of populus euphratica based on BSA mixed pool sequencing analysis - Google Patents

Specific DNA molecular marker for sex identification of populus euphratica based on BSA mixed pool sequencing analysis Download PDF

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CN112430680B
CN112430680B CN202011213071.1A CN202011213071A CN112430680B CN 112430680 B CN112430680 B CN 112430680B CN 202011213071 A CN202011213071 A CN 202011213071A CN 112430680 B CN112430680 B CN 112430680B
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populus euphratica
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李志军
吴智华
张山河
刘虹
曲文蕊
覃瑞
翟军团
韩晓莉
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Tarim University
South Central Minzu University
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Abstract

The invention develops a specific DNA molecular marker for sex identification of populus euphratica based on BSA mixed pool sequencing analysis, and designs a corresponding detection primer based on sequence information of the specific molecular marker, so that the sex of populus euphratica from different places can be economically, rapidly, accurately and sensitively distinguished, technical support is provided for the selective transplanting of female plants or male plants of populus euphratica in nursery stock breeding, and theoretical support is also provided for the popularization of popularizing the populus euphratica in desertification control.

Description

Specific DNA molecular marker for sex identification of populus euphratica based on BSA mixed pool sequencing analysis
Technical Field
The invention relates to the technical field of biological information and biotechnology, in particular to a aspen BSA mixed pool sequencing analysis and development of specific DNA molecular markers for aspen sex identification.
Background
The populus euphratica (Populus euphratica Oliv.) is an important arbor species in desert areas, is mainly distributed in northwest China, has the most wide distribution in Xinjiang, has stronger drought resistance, cold resistance and salt and alkali resistance, and has irreplaceable effects in the aspects of maintaining the balance of desert ecosystem, restraining desert expansion, protecting biodiversity, guaranteeing ecological safety in industrial and agricultural production and the like. In Xinjiang, populus euphratica is often the first tree species for urban landscaping and protective forest construction. In recent years, due to the influence of natural and artificial factors such as agricultural reclamation, wood harvesting, unreasonable water resource development and the like, large-area natural populus euphratica forests, ash She Huyang forests decline, are not firm and even die, and populus euphratica is listed as a third-level gradually-endangered protection plant in China.
Populus euphratica belongs to hermaphroditic plants, and the male aspen is the first choice for constructing a pavement tree or a protective forest because the trunk nature and the growth vigor of the male aspen are better than those of the female aspen, and the female aspen brings great inconvenience to pedestrians after 7-9 months of seed scattering. However, the populus euphratica seedlings can enter the stage of flowering and fruiting after taking 8-10 years, and the sex of the populus euphratica seedlings is difficult to accurately identify before flowering. The application of the populus euphratica male plants in urban landscaping and protective forest construction is greatly hindered.
The mixed packet analysis (Bulked Segregant Analysis, BSA) first proposed by Michlcore et al is a practical gene marker localization technique for identifying genomic regions containing genetic loci affecting a trait, and combining deep sequencing can effectively accelerate the gene localization process. The rationale is that markers linked to traits will be polymorphic between the two extreme pools, whereas markers that are far from the target gene or that are not linked will exhibit random heterozygosity between the two extreme pools. The method can rapidly obtain the molecular marker linked with the character. Is generally used for positioning quality trait genes or quantitative trait loci controlled by a few major QTLs. With the rapid development of new generation sequencing technologies, new strategies utilizing BSA and NGS to assist in high-throughput genotyping have been proposed.
By the present invention, although reports about differences in leaf anatomy and isozymes between male and female populus strains have been made, they are insufficient as a basis for early sex identification of populus seedlings. With the development of molecular biology, the DNA molecular marker technology becomes an accurate and reliable method in the early sex identification of hermaphrodite plants. In populus plants, only the patent with the authority of CN201911012229.6 discloses two DNA molecular markers specific to female for early sex identification of populus alba, but the development and application of related molecular markers for sex identification of populus euphratica are not seen.
Disclosure of Invention
In order to solve the problem of rapid and accurate identification of the sex of populus euphratica by utilizing reliable DNA molecular markers, the invention develops specific DNA molecular markers for sex identification of populus euphratica based on BSA mixed pool sequencing analysis, designs corresponding detection primers based on sequence information of the specific molecular markers, and achieves the aim of rapidly, accurately and sensitively distinguishing the sex of populus euphratica from different places.
In one embodiment, the invention provides a molecular marker for identifying the sex of populus diversifolia, which is characterized in that the molecular marker is obtained by amplifying the following primer pairs, wherein the primer pairs are selected from the group consisting of: SEQ ID NO.7 and SEQ ID NO.8; SEQ ID NO.9 and SEQ ID NO.10; SEQ ID NO.11 and SEQ ID NO.12; SEQ ID NO.13 and SEQ ID NO.14. The molecular marker is contained only in male populus euphratica.
Preferably, the molecular marker further comprises a control fragment, characterized in that the control fragment is selected from the group consisting of SEQ ID No.1 and SEQ ID No.2; SEQ ID NO.3 and SEQ ID NO.4; SEQ ID NO.5 and SEQ ID NO. 6; the control fragment is contained in both female and male populus euphratica.
More preferably, the molecular marker is amplified by a primer set of SEQ ID NO.7-8; SEQ ID NO.9-10; SEQ ID NO.11-12; SEQ ID NOS.13-14.
Further preferably, the molecular marker control fragment is obtained by amplifying a primer set shown in SEQ ID NO.1-2; SEQ ID NO.3-4; SEQ ID NO.5-6.
In one embodiment, the invention provides an application of a molecular marker or a detection primer in detecting sexes of populus euphratica, preferably, the detection method is that PCR amplification is carried out by using a primer pair, and on the basis of amplifying a strip by using a common marker of male and female, if a strip with a male characteristic marker with a size of 100-300bp is amplified, the strip is male; if the target band is not amplified by the male-labeled primer pair, the primer pair is female.
In one embodiment, the inventionThe method for detecting the sex of the female and male of the populus euphratica is characterized in that genome DNA of the populus euphratica is used as a template, the primer pair of claim 3 is used for PCR amplification, and on the basis of amplifying a band by the common male and female markers, if a band with a male characteristic marker with the size of 100-300bp is amplified, the band is male; if the target band is not amplified by the male-labeled primer pair, the primer pair is female. Preferably, the PCR amplification system is 2X Taq PCR Master Mix (Vazyme) 12.5. Mu.L, 1. Mu.L (10. Mu. Mol/L) of each of the upstream and downstream primers, 1. Mu.L of the template, and ddH 2 O is added to 25 mu L;
compared with the prior art, the invention has the following advantages:
the marking and detecting flow developed by the invention is that 10 aspen from different places are detected, and the detecting efficiency is found to be 100%. The invention can economically, rapidly, accurately and sensitively distinguish the sexes of the populus euphratica from different places, provides technical support for the selective transplanting of female plants or male plants in the breeding of the seedlings of the populus euphratica and also provides theoretical support for the popularization of the popularizing work of the populus euphratica in desertification control.
Drawings
Fig. 1: the invention uses a primer pair of a common female and male mark F1 to detect the sex identification of populus euphratica;
fig. 2: the invention uses a primer pair of a common female and male mark F2 to detect the sex identification of populus euphratica;
fig. 3: the invention uses a primer pair of a common female and male mark F3 to detect the sex identification of populus euphratica;
fig. 4: the invention uses a primer pair of a DNA molecular marker M1 specific to male to detect the sex identification of populus euphratica;
fig. 5: the invention uses a primer pair of a DNA molecular marker M2 specific to male to detect the sex identification of populus euphratica;
fig. 6: the invention uses a primer pair of a DNA molecular marker M3 specific to male to detect the sex identification of populus euphratica;
fig. 7: the invention uses a primer pair of a DNA molecular marker M4 specific to male to detect the identification of the sex of populus euphratica. And (3) injection: m in fig. 1-7: a Marker; lanes Populus euphratica (female) 1-10: 10 aspen female samples from different sites; populus euphratica, 1-10: 10 aspen male plant samples from different sites.
Detailed Description
In order to better understand the technical scheme provided by the invention, the technical scheme provided by the invention is described in detail below with reference to the embodiment.
EXAMPLE 1 Assembly of Male and female Populus euphratica genomes
High quality genomic DNA of male and female aspen leaves was extracted and libraries were constructed for Illumina, pacBio and Hi-C sequencing, respectively. And performing preliminary assembly on the reads obtained based on PacBio sequencing, and performing error correction on the assembly result by using the reads obtained by Illumina sequencing to obtain a genome of a sketch version. Assembling the male and female genomes by using a Hi-C sequencing technology, annotating and evaluating the male and female genomes, and obtaining a high-quality male and female genome sequence at the chromosome level.
Example 2 development of a candidate DNA molecular marker related to populus euphratica gender:
and (3) carrying out correlation analysis on BSA-seq data of 96 female plants and 97 male plants by taking a male genome as a reference to obtain a region with the female pool depth of 0 continuously and the male pool depth of more than 10 continuously. Sequences including 150bp upstream and downstream were extracted from these regions as candidate sex markers. Blast is performed on the genome of the obtained markers and assembled female strains, and the obtained 30 candidate markers are initially classified into 16 markers for male and female, 10 markers for male and female possibly, and 4 markers for male.
Example 3 design of sex marker candidate primer and marker screening
Primer 5 software is adopted to design primers by taking 30 candidate sex marks as target fragments, the length of the primers is 18-22bp, and the GC% is 35-48%. PCR verification screening is carried out on the corresponding markers by using 30 pairs of primers and populus female and male plant genome DNA as templates, and finally, the optimal 3 male and female common markers F1-F3 are obtained from 16 common markers of male and female, and 4 male specific markers M1-M4 are obtained from 14 possible common markers of male and female and specific markers of male, and are specifically shown in table 1.
TABLE 1 sex markers of populus euphratica and primer sequences thereof
Example 4 sex identification of Populus euphratica
1. Experimental materials
10 female and male populus euphratica strains with known sexes from different places are shown in table 2.
Table 2 aspen samples from 10 sites
2. Experimental method
PCR amplification detection is carried out on the populus euphratica sample (table 2) by utilizing 3 primer pairs of DNA molecular markers which are preferably used for both male and female in the development mark of the patent and 4 primer pairs of DNA molecular markers which are special for male (table 1);
the amplification system was 2X Taq PCR Master Mix (Vazyme) 12.5. Mu.L, 1. Mu.L (10. Mu. Mol/L) of each of the upstream and downstream primers, 1. Mu.L of the template, and ddH was added 2 O is added to 25 mu L;
the PCR amplification procedure was 95℃pre-denatured for 3min; denaturation at 95℃for 15s, annealing at 58℃for 15s, extension at 72℃for 1min, and cycling for 35 times; extending at 72 ℃ for 10min;
the PCR product is taken 5 mu L and is subjected to electrophoresis detection by agarose gel with the concentration of 1%, and the result shows that 3 marked primer pairs F1-F3 shared by the male and the female can amplify target bands (figures 1-3) in all samples, meanwhile, 4 marked primer pairs M1-M4 special for the male can amplify target bands (figures 4-7) of 100-300bp special for the male, and a female sample has no bands, so that 7 DNA molecular markers can be jointly used for sex identification of the populus euphratica.
3. Experimental results
The labeling and detection procedures developed in the present invention were carried out by examining 10 strains of female and male populus euphratica from different sites with known sexes (Table 2), and found that the detection efficiency was 100% (FIGS. 1-7).
While the invention has been described in detail with respect to the general description and specific embodiments thereof, it will be apparent to those skilled in the art that various modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
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Claims (6)

1. A set of molecular markers for identifying the sex of populus diversifolia, characterized in that the molecular markers consist of a male specific marker and a male-female common marker, the male specific marker is obtained by amplifying the genome of populus diversifolia by a primer set a, and the primer set a is selected from the group consisting of: SEQ ID NO.7 and SEQ ID NO.8; SEQ ID NO.9 and SEQ ID NO.10; at least one of SEQ ID NO.11 and SEQ ID NO.12 or SEQ ID NO.13 and SEQ ID NO.14, wherein the male and female common markers are obtained by amplifying the populus euphratica genome by the primer group B; the primer group B is selected from the group consisting of: SEQ ID NO.1-2; at least one of SEQ ID NO.3-4 or SEQ ID NO.5-6.
2. The molecular marker according to claim 1, wherein the primer set a is SEQ ID No.7-8; SEQ ID NO.9-10; SEQ ID No.11-12 and SEQ ID No.13-14.
3. The molecular marker according to claim 1, wherein the primer set B is SEQ ID No.1-2; SEQ ID No.3-4 and SEQ ID No.5-6.
4. A kit for detecting the sex of populus diversifolia, which comprises the primer set a and the primer set B as set forth in claim 1.
5. Use of the molecular marker of any one of claims 1-3 for detecting male and female sex of populus euphratica.
6. A method for detecting the sex of female and male populus, which is characterized in that populus genome DNA is used as a template, the primer group A and the primer group B in claim 1 are used for carrying out PCR amplification on the populus genome DNA, and on the basis of amplifying a band by a common male and female marker, if a band of a male specific marker with the size of 100-300bp is amplified, the band is male; if the target band is not amplified by the male-labeled primer pair, the primer pair is female.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108754015A (en) * 2018-06-28 2018-11-06 杭州师范大学 Specific molecular marker PCMI-F001 for Cathay poplar female seedling Rapid identification
CN109724976A (en) * 2018-12-21 2019-05-07 塔里木大学 Trees Sex judging method based on paper chromatography
CN111269974A (en) * 2020-03-09 2020-06-12 南京林业大学 Specific genomic DNA sequence of male Populus microphylla strain and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108754015A (en) * 2018-06-28 2018-11-06 杭州师范大学 Specific molecular marker PCMI-F001 for Cathay poplar female seedling Rapid identification
CN109724976A (en) * 2018-12-21 2019-05-07 塔里木大学 Trees Sex judging method based on paper chromatography
CN111269974A (en) * 2020-03-09 2020-06-12 南京林业大学 Specific genomic DNA sequence of male Populus microphylla strain and application thereof

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